The present invention relates to acylase immobilizates on the basis of chemically functionalized celluloses, and the preparation thereof.
The term "acylases," as used herein, refers to N-acyl-L-amino acid amidohydrolases. These are water-soluble proteins of various provenance. Preferred sources for them are either animal organs, such as pig kidneys and the pancreases of cattle, or special strains of microorganisms, such as Aspergillus orycae. Depending on their origin, the acylases have different substrate specificities, in that a particular species has a special facility for splitting off a particular acyl group, such as the acetyl group, for example. What is common to all acylases, however, is their ability to perform a selective deacetylation of N-acyl-L-amino acids.
The stereospecific enzymatic hydrolysis of the bond between the amino group and the acyl radical can be utilized technically for the production of L-amino acids by separating the L-amino acid liberated in the asymmetrical hydrolysis, racemizing the N-acyl-D form that has remained intact, again performing an enzymatic cleavage of the N-acyl-L part of the racemate, and pursuing the process until the N-acyl form has been used up. In this manner it is possible to transform the N-acyl racemate of the particular amino acid to the desired levorotatory form. The cleavage is accomplished under very mild conditions which cost little as regards energy input: the pH values of the substrates being cleaved are usually close to neutral, for example. The cleavage is performed as a rule at temperatures within or close to the physiological range. On account of these advantageous properties of the acylases, there is a great interest in using them to achieve an especially economical separation of the optical antipodes of amino acids on a technical scale.
The solubility of the acylases, however, is an obstacle to their technical utilization. The enzymes, which produce their action even in very low concentrations, have to be recovered to the greatest possible extent from large volumes of substrate, since they are expensive and therefore have to be reused until their enzymatic activity is exhausted.
It is necessary to remove the acylases from the cleavage solutions also in order to free them from the proteins and their soluble by-products and decomposition products. The solubility, great dilution and the chemical similarity of the acylases to their accompanying substances are obstacles, however, to an economically acceptable recovery and they considerably complicate any handling of the acylases.
Therefore, it was possible to expect insolubilized acylases to offer advantages in a biotechnology such as the enzymatic resolution of racemates: the immobilizates can be separated from the cleavage solutions by simple physical separating methods such as filtration and centrifugation, and can be recovered for reuse. They can be put into reactors, such as reactor columns for example, in the form of solid, stationary phases, which is an especially desirable arrangement for the continuous performance of the optical cleavage. Furthermore, enzymes fixed on carriers are often more stable thermally than the soluble forms, they permit in some cases operation at higher temperatures to increase the volume-time yield of the process, and they are often less sensitive to denaturing influences.
Consequently, enzymes made insoluble by applying them to carrier materials have already been proposed.
The carriers which have hitherto been proposed for the insolubilization of cleavage-active biogenic proteins, however, do not satisfy to the desired degree the requirements which must be met by the immobilizates used in an industrial process. The reasons for this are as follows:
Inorganic carriers of a mainly hydrophobic character, such as porous glasses for example, do have better mechanical properties than many hydrophilic organic carriers. The binding capacity of inorganic carriers for the bioactive proteins, however, is very limited. In contact with the neutral to weakly alkaline substrate solutions, the enzyme activity diminishes so greatly that these immobilizates are unsuitable for technical application. Also, such carrier materials often have excessively high solubilities in the substrate solutions; the result is poor stability of the fixed enzyme and contamination of the cleavage solutions. Hydrophilic carriers, such as modified polysaccharides or polyacrylamide gels, prove to have very undesirable, sometimes irreversible swelling phenomena, and often they have porosity caused by swelling, so that a rapid transformation of the substrates is impossible due to interference with the diffusion. The swelling phenomena are especially disadvantageous when the gel-like immobilizates are used in solid-bed reactors. Acylase adsorptively fixed to DEAE Sephadex, a dextran provided with diethylaminoethyl groups, or to DEAE cellulose, does not adhere strongly enough to achieve sufficient half-value times for their hydrolytic activity. The polar bond content developed in the formation of such immobilizates does not attain the strength and insensitivity to milieu factors, such as internal concentration and internal charge, of covalently bound enzymes.
The use of cellulose or cellulose derivatives as carriers has been impeded generally by the fact that cellulose is insoluble without chemical alteration, and the reactions performed in suspended phase require drastic conditions, and lead to no more than partial reactions on account of the tangling of the cellulose chains.
Chemically modified cellulose, such as alkali celluloses, can be functionalized with only a few reagents and are hard to free from the harmful alkalies. Immobilizates on the basis of the known modified celluloses are also easily oxidizable and have to be handled with the exclusion of air.